Abstract
Osteoblast differentiation from mesenchymal cells is regulated by multiple signalling pathways. Here we have analysed the roles of Fibroblast Growth Factor (FGF) and canonical Wingless-type MMTV integration site (Wnt/β-Catenin) signalling pathways on zebrafish osteogenesis. We have used transgenic and chemical interference approaches to manipulate these pathways and have found that both pathways are required for osteoblast differentiation in vivo. Our analysis of bone markers suggests that these pathways act at the same stage of differentiation to initiate expression of the osteoblast master regulatory gene osterix (osx). We use two independent approaches that suggest that osx is a direct target of these pathways. Firstly, we manipulate signalling and show that osx gene expression responds with similar kinetics to that of known transcriptional targets of the FGF and Wnt pathways. Secondly, we have performed ChIP with transcription factors for both pathways and our data suggest that a genomic region in the first intron of osx mediates transcriptional activation. Based upon these data, we propose that FGF and Wnt/β-Catenin pathways act in part by directing transcription of osx to promote osteoblast differentiation at sites of bone formation.
Highlights
The bony skeleton initially develops in one of two ways, either by ossification of a cartilage template or in the absence of a cartilage template
Activation of Fibroblast Growth Factor (FGF) signalling in vitro results in reduced expression of Alkaline phosphatase (ALP) and Col1 and induces osteoblast apoptosis [18,19,20]. These results suggest that FGF signalling may play different roles during osteoblast differentiation and that timing and strength of the FGF signal is crucial in these outcomes
FGF signalling is required for ossification and the development of mature osteoblasts To determine whether FGF signalling acts during zebrafish bone development we took advantage of a zebrafish transgenic line called hs:dnfgfr1 that expresses a dominant negative form of the FGF under control of a heat shock promoter (Tg(hsp70l:dnfgfr1-EGFP); [52])
Summary
The bony skeleton initially develops in one of two ways, either by ossification of a cartilage template (chondral ossification) or in the absence of a cartilage template (achondal ossification). Osteoblasts (specialised cells that synthesise bone) are derived from multipotent mesenchymal stem cells which are found in different tissues. Osteoblasts initially differentiate in the perichondrium (a tissue which surrounds the cartilage), and in achondral bone development osteoblasts differentiate in mesenchymal cell condensations. Later on in development and in adults, osteoblast progenitors are found in the bone marrow as well as in the periosteum (a tissue which surrounds bone). Genetic analysis in mice has identified two transcription factors, Runx (Runt-related transcription factor 2) and Osx ( known as Sp7), that act in a transcriptional cascade during osteoblast differentiation.
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